Philippe, J. | Search bioRxiv

bioRxiv2023

Structure and activity of botulinum neurotoxin X

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 [A] resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

biochemistry↗

bioRxiv2019

Gclc deletion in surface-ectoderm tissues induces microphthalmia

Glutamate cysteine ligase catalytic subunit (Gclc) is the catalytic subunit for the glutamate-cysteine ligase (Gcl) enzyme. Gcl catalyzes the rate limiting step in glutathione (GSH) synthesis. Gclc is highly expressed in the developing eye. To define the regulatory role of Gclc in eye development, we developed a novel, Le-Cre transgene-driven, Gclc knockout mouse model. Gclcf/f/Le-CreTg/- mice present with deformation of the retina, cornea, iris, and lens, consistent with a microphthalmia phenotype. Controlling for the microphthalmia phenotype of Gclcwt/wt/Le-CreTg/- mice revealed that Gclcf/f/Le-CreTg/- mice have a more severe microphthalmia phenotype. Thus, the loss of Gclc expression exacerbates the microphthalmia phenotype in Le-Cre mice. Gclcf/f/Le-CreTg/- eyes present with reduced retinal and lens epithelium proliferation and increased lens cell death. Imaging mass spectrometry of ocular tissues revealed changes in the intensity and distribution of several lipid species and proteins in the retina and corneas of Gclcf/f/Le-CreTg/- eyes. Lastly, using splice-blocking morpholinos and CRISPR/Cas9, we created two gclc knockdown zebrafish models, both of which display a microphthalmia phenotype. Combined, the mouse and zebrafish results indicate that, in chordates, Gclc has a conserved role in regulating eye development. In summary, these novel animal models are useful tools for elucidating the mechanisms involved in microphthalmia development.

developmental biology↗